Human plasminogen is a single-chain protein containing 791 amino acid residues. Activation of plasminogen to plasmin results from a single cleavage of the Arg561-Val562 peptide bond in the zymogen. The resulting plasmin molecule is a two-chain, disulfide-linked serine protease with trypsin-like specificity (cleaves after Lys and Arg). The amino-terminal heavy chain of plasmin (residues 1-561, ˜60 kDa) is composed of five kringle domains, each containing approximately 80 amino acid residues. The kringle domains are responsible for the regulatory properties of plasminogen, such as interaction with activation inhibitors, e.g., Cl−1 ions; with activation stimulators, e.g., ε-aminocaproic, acid; with mammalian and bacterial cells; and with other proteins, such as the plasmin physiological substrate, fibrin and plasmin inhibitor, α2-antiplasmin. Of all five kringles, kringle 1 is one of the most multi-functional: its lysine-binding activity has been shown to be responsible for plasmin interaction with α2-antiplasmin and fibrin. See Wiman, B., et al., Biochim. Biophys. Acta 579: 142-154 (1979); and Lucas, M. A., et al., J. Biol. Chem. 258: 4249-4256′(1983).
The C-terminal light chain of plasmin (residues 562-791, ˜25 kDa) is a typical serine protease, homologous to trypsin and containing the classic serine protease catalytic triad: His603, Asp646 and Ser741. Plasminogen contains 24 disulfide bridges and 2 glycosylation sites on Asn289 and Thr346.
The limited proteolysis of plasminogen by elastase has been shown to result in three major fragments (Sottrup-Jensen, L., et al., Prog. Chem. Fibrinol. Thrombol. 3: 191-209 (1978)). First fragment, K1-3, includes the first three kringles and can be isolated in two versions, Tyr80-Val338 and Tyr80-Val354. The second fragment, K4, corresponds to the fourth kringle and includes residues Val355-Ala440. The last, C-terminal fragment (the so-called mini-plasminogen) includes residues Val443-Asn791 and consists of the fifth kringle and the serine protease domain. Mini-plasminogen can be activated in the same way as plasminogen, forming mini-plasmin.
Because of the complex structure of the full-length plasminogen molecule, bacterial expression systems have not proven useful for recombinant plasminogen production. Plasminogen is produced in the form of insoluble inclusion bodies and is not re-foldable from that state. Further, the expression of plasminogen in mammalian cells is complicated by intracellular activation of plasminogen into plasmin and the resulting cytotoxicity. Production of fully active plasminogen using insect cells is possible, however, this system is not suitable for large-scale production due to low yield. Further, as with any recombinant protein scheme, the potential exists for encountering immunogenicity problems in the subject receiving the therapeutic recombinant protein.
Immunogenicity can be a barrier to the effective and/or efficient utilization of certain recombinant protein therapeutic schemes. Immunogenicity is a complex series of responses to a substance (e.g., the chemical structure of a protein including the amino acid sequence) that is perceived as foreign and may include production of neutralizing and non-neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation, and anaphylaxis. Immunogenicity may limit the efficacy and safety of a protein therapeutic in multiple ways. Efficacy can be reduced directly by the formation of neutralizing antibodies. Efficacy may also be reduced indirectly, as binding to either neutralizing or non-neutralizing antibodies typically leads to rapid clearance from serum. Severe side effects and even death may occur when an immune reaction is raised. One special class of side effects results when neutralizing antibodies cross-react with an endogenous protein and block its function.
Accordingly, a modified recombinant protein, possessing the desirable characteristics (e.g., regions with native-like chemical structures) of plasmin/plasminogen while lacking certain negative characteristics and being capable of production in recombinant protein expression systems including bacterial cells in substantial quantities, is desirable.